Alcohol (ethanol, EtOH) consumption exhibits a wide variety of effect on the human body, ranging from beneficial effect on the cardiovascular system to detrimental effects on other systems, including suppression of the immune system. The mechanisms leading to such versatile effect of EtOH are largely unknown. The goal of this proposal is to define the influence of EtOH on molecular mechanisms involved in regulation of Ca2+-dependent LPS-triggered macrophage (Mf) activation. Our preliminary results suggest that EtOH inhibits pathogen-mediated Mf activation, specifically, upon acute EtOH exposure Mf produce less pro-inflammatory cytokine TNF? when a bacterial-derived product, lipopolysaccharide (LPS) triggers cell activation via Toll-like receptor 4 (TLR4). We provide novel preliminary data that engagement of TLR4 with LPS triggers rapid increase in cytoplasmic Ca2+ in Mf. Moreover, Ca2+ depletion or chemical block of Ca2+ influx abolished LPS-induced TNF? production in Mf. EtOH impairs LPS-induced phosphorylation of CaMKII, a Ca2+-dependent kinase with key role in assembly of initial TLR4 signaling complex. EtOH stimulates Ca2+-dependent activation of NF-AT, a nuclear factor that regulates TNF? production. Based on our preliminary data, we postulated that Ca2+ plays a key role in TLR4-mediated Mf activation and second, EtOH-induced impairment of Ca2+-mediated signaling in Mf. We hypothesize that a) EtOH- conditioned modulation of Ca2+ in the cytoplasm leads to defective signaling via initial TLR4 signaling complex, and/or b) alcohol modulates the previously unknown/under-estimated LPS-triggered Ca2+-calmodulin-calcineurin-CaMKII-NF-AT pathway. We hypothesized that the mechanisms of the acute EtOH-conditioned impairment of LPS-induced Mf activation include alterations of Ca2+ homeostasis and induce modulation of large conductance Ca2+ activated potassium channels. This proposal addresses our hypothesis to unravel the role of Ca2+ in EtOH-induced impairment of TLR4-mediated innate immune activation. PUBLIC HEALTH RELEVANCE: Alcohol consumption exhibits a wide variety of effect on human body, ranging from beneficial effect on cardiovascular system to detrimental effects on all other system, including suppression of the immune system. The mechanisms leading to such versatile effect of EtOH are largely unknown. The overall goal of this research proposal is to define the influence of alcohol on the molecular mechanisms involved in regulation macrophage activation. Macrophages (Mf) produce pro-inflammatory cytokine TNF? when a bacterial- derived product, lipopolysaccharide (LPS) binds to the Toll-like receptor 4 (TLR4). Alcohol inhibits LPS-induced TNF? production. We provide novel preliminary data that engagement of TLR4 increases the cytoplasmic Ca2+ in Mf. More importantly and similar to acute alcohol, Ca2+ depletion or chemical block abolished the LPS-induced TNF? production. Here we postulated that, first; Ca2+ plays a key role in TLR4-mediated Mf activation and second, alcohol modulates the Ca2+ homeostasis in Mf. We hypothesize that a) alcohol-conditioned modulation of Ca2+ leads to defective formation of the initial TLR4 signaling complex, and/or b) alcohol modulates the previously unknown/under-estimated LPS-triggered Ca2+-calmodulin-calcineurin-CaMKII-NFAT pathway. We further hypothesized that acute alcohol modulates the Ca2+-dependent large conductance Ca2+ activated potassium channels. This proposal will address our hypothesis in order to unravel the role of Ca2+ in EtOH-induced impairment of TLR4-mediated innate immune activation.